Sustained oral spermidine supplementation rescues functional and structural defects in COL6-deficient myopathic mice.

COL6 (collagen type VI)-related myopathies (COL6-RM) are a distinct group of inherited muscle disorders caused by mutations of COL6 genes and characterized by early-onset muscle weakness, for which no cure is available yet. Key pathophysiological features of COL6-deficient muscles involve impaired macroautophagy/autophagy, mitochondrial dysfunction, neuromuscular junction fragmentation and myofiber apoptosis. Targeting autophagy by dietary means elicited beneficial effects in both col6a1 null (col6a1-/-) mice and COL6-RM patients. We previously demonstrated that one-month per os administration of the nutraceutical spermidine reactivates autophagy and ameliorates myofiber defects in col6a1-/- mice but does not elicit functional improvement. Here we show that a 100-day-long spermidine regimen is able to rescue muscle strength in col6a1-/- mice, with also a beneficial impact on mitochondria and neuromuscular junction integrity, without any noticeable side effects. Altogether, these data provide a rationale for the application of spermidine in prospective clinical trials for COL6-RM.Abbreviations: AChR: acetylcholine receptor; BTX: bungarotoxin; CNF: centrally nucleated fibers; Colch: colchicine; COL6: collagen type VI; COL6-RM: COL6-related myopathies; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; NMJ: neuromuscular junction; Spd: spermidine; SQSTM1/p62: sequestosome 1; TA: tibialis anterior; TOMM20: translocase of outer mitochondrial membrane 20; TUNEL: terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling.

Ambra1 deficiency impairs mitophagy in skeletal muscle.

BACKGROUND: Maintaining healthy mitochondria is mandatory for muscle viability and function. An essential surveillance mechanism targeting defective and harmful mitochondria to degradation is the selective form of autophagy called mitophagy. Ambra1 is a multifaceted protein with well-known autophagic and mitophagic functions. However, the study of its role in adult tissues has been extremely limited due to the embryonic lethality caused by full-body Ambra1 deficiency. METHODS: To establish the role of Ambra1 as a positive regulator of mitophagy, we exploited in vivo overexpression of a mitochondria-targeted form of Ambra1 in skeletal muscle. To dissect the consequence of Ambra1 inactivation in skeletal muscle, we generated muscle-specific Ambra1 knockout (Ambra1fl/fl :Mlc1f-Cre) mice. Mitochondria-enriched fractions were obtained from muscles of fed and starved animals to investigate the dynamics of the mitophagic flux. RESULTS: Our data show that Ambra1 has a critical role in the mitophagic flux of adult murine skeletal muscle and that its genetic inactivation leads to mitochondria alterations and myofibre remodelling. Ambra1 overexpression in wild-type muscles is sufficient to enhance mitochondria clearance through the autophagy-lysosome system. Consistently with this, Ambra1-deficient muscles display an abnormal accumulation of the mitochondrial marker TOMM20 by +76% (n = 6-7; P < 0.05), a higher presence of myofibres with swollen mitochondria by +173% (n = 4; P < 0.05), and an alteration in the maintenance of the mitochondrial membrane potential and a 34% reduction in the mitochondrial respiratory complex I activity (n = 4; P < 0.05). Lack of Ambra1 in skeletal muscle leads to impaired mitophagic flux, without affecting the bulk autophagic process. This is due to a significantly decreased recruitment of DRP1 (n = 6-7 mice; P < 0.01) and Parkin (n = 6-7 mice; P < 0.05) to the mitochondrial compartment, when compared with controls. Ambra1-deficient muscles also show a marked dysregulation of the endolysosome compartment, as the incidence of myofibres with lysosomal accumulation is 20 times higher than wild-type muscles (n = 4; P < 0.05). Histologically, Ambra1-deficient muscles of both 3- and 6-month-old animals display a significant decrease of myofibre cross-sectional area and a 52% reduction in oxidative fibres (n = 6-7; P < 0.05), thus highlighting a role for Ambra1 in the proper structure and activity of skeletal muscle. CONCLUSIONS: Our study indicates that Ambra1 is critical for skeletal muscle mitophagy and for the proper maintenance of functional mitochondria.

Autophagy in the mesh of collagen VI.

Autophagy is a very versatile process through which the cell degrades damaged long-lived proteins, entire organelles, or pathogens, by engulfing them in characteristic double-membrane vesicles and conveying the cargo to lysosomes. It is a dynamic pathway tunable at multiple levels and responsive to nutrient and stress stimuli, also coming from the extracellular microenvironment and its remodeling. In the extracellular matrix, collagen type VI forms a distinctive set of beaded microfilaments that assemble into an intricate and multimodular meshwork of tightly linked proteins and surface receptors. When missing or defective, collagen VI triggers a series of pathological events in skeletal muscle and other tissues, with a remarkable impact on key cell processes, such as apoptosis and autophagy. In this review, we discuss the current knowledge about collagen VI regulation of autophagy in the different experimental models and human pathologies where it was studied, and provide some hints for future directions aimed at the fine dissection of this intriguing relationship, as well as its prospective translational impact for disease and therapy.

Role of adiponectin in the metabolism of skeletal muscles in collagen VI-related myopathies.

The role of adiponectin has been particularly deepened in diabetic muscles while the study of adiponectin in hereditary myopathies has been marginally investigated. Here, we report the study about adiponectin effects in Col6a1-/- (collagen VI-null) mice. Col6a1-/- mice show myophatic phenotype closer to that of patients with Bethlem myopathy, thus representing an excellent animal model for the study of this hereditary disease. Our findings demonstrate that Col6a1-/- mice have decreased plasma adiponectin content and diseased myoblasts have an impaired autocrine secretion of the hormone. Moreover, Col6a1-/- myoblasts show decreased glucose uptake and mitochondria with depolarized membrane potential and impaired functionality, as supported by decreased oxygen consumption. Exogenous addition of globular adiponectin modifies the features of Col6a1-/- myoblasts, becoming closer to that of the healthy myoblasts. Indeed, globular adiponectin enhances glucose uptake in Col6a1-/- myoblasts, modifies mitochondrial membrane potential, and restores oxygen consumption, turning closer to those of wild-type myoblasts. Finally, increase of plasma adiponectin level in Col6a1-/- mice is induced by fasting, a condition that has been previously shown to lead to the amelioration of the dystrophic phenotype. Collectively, our results demonstrate that exogenous replenishment of adiponectin reverses metabolic abnormalities observed in Col6a1-/- myoblasts. KEY MESSAGES: Col6a1-/- mice have decreased level of plasma adiponectin. Myoblasts from Col6a1-/- muscles have impaired local adiponectin secretion. Col6a1-/- myoblasts reveal altered metabolic features. Addition of exogenous adiponectin ameliorates Col6a1-/- metabolic features.

Extracellular Collagen VI Has Prosurvival and Autophagy Instructive Properties in Mouse Fibroblasts.

Collagen VI (ColVI) is an abundant and distinctive extracellular matrix protein secreted by fibroblasts in different tissues. Human diseases linked to mutations on ColVI genes are primarily affecting skeletal muscle due to non-cell autonomous myofiber defects. To date, it is not known whether and how fibroblast homeostasis is affected by ColVI deficiency, a critical missing information as this may strengthen the use of patients' fibroblasts for preclinical purposes. Here, we established primary and immortalized fibroblast cultures from ColVI null (Col6a1-/-) mice, the animal model of ColVI-related diseases. We found that, under nutrient-stringent condition, lack of ColVI affects fibroblast survival, leading to increased apoptosis. Moreover, Col6a1-/- fibroblasts display defects in the autophagy/lysosome machinery, with impaired clearance of autophagosomes and failure of Parkin-dependent mitophagy. Col6a1-/- fibroblasts also show an increased activation of the Akt/mTOR pathway, compatible with the autophagy impairment, and adhesion onto purified ColVI elicits a major effect on the autophagic flux. Our findings reveal that ColVI ablation in fibroblasts impacts on autophagy regulation and cell survival, thus pointing at the new concept that this cell type may contribute to the pathological features of ColVI-related diseases.

Autophagy activation in COL6 myopathic patients by a low-protein-diet pilot trial.

A pilot clinical trial based on nutritional modulation was designed to assess the efficacy of a one-year low-protein diet in activating autophagy in skeletal muscle of patients affected by COL6/collagen VI-related myopathies. Ullrich congenital muscular dystrophy and Bethlem myopathy are rare inherited muscle disorders caused by mutations of COL6 genes and for which no cure is yet available. Studies in col6 null mice revealed that myofiber degeneration involves autophagy defects and that forced activation of autophagy results in the amelioration of muscle pathology. Seven adult patients affected by COL6 myopathies underwent a controlled low-protein diet for 12 mo and we evaluated the presence of autophagosomes and the mRNA and protein levels for BECN1/Beclin 1 and MAP1LC3B/LC3B in muscle biopsies and blood leukocytes. Safety measures were assessed, including muscle strength, motor and respiratory function, and metabolic parameters. After one y of low-protein diet, autophagic markers were increased in skeletal muscle and blood leukocytes of patients. The treatment was safe as shown by preservation of lean:fat percentage of body composition, muscle strength and function. Moreover, the decreased incidence of myofiber apoptosis indicated benefits in muscle homeostasis, and the metabolic changes pointed at improved mitochondrial function. These data provide evidence that a low-protein diet is able to activate autophagy and is safe and tolerable in patients with COL6 myopathies, pointing at autophagy activation as a potential target for therapeutic applications. In addition, our findings indicate that blood leukocytes are a promising noninvasive tool for monitoring autophagy activation in patients.

Lack of collagen VI promotes neurodegeneration by impairing autophagy and inducing apoptosis during aging.

Collagen VI is an extracellular matrix (ECM) protein with a broad distribution in different tissues and mostly deposited at the close periphery of the cell surface. Previous studies revealed that collagen VI protects neurons from the toxicity of amyloid-ßpeptides and from UV-induced damage. However, the physiological role of this protein in the central nervous system (CNS) remains unknown. Here, we established primary neural cultures from murine cortex and hippocampus, and carried out in vitro and in vivo studies in wild-type and collagen VI null (Col6a1-/-) mice. Col6a1-/- neural cultures displayed an increased incidence of spontaneous apoptosis and higher vulnerability to oxidative stress, accompanied by altered regulation of autophagy with increased p62 protein levels and decreased LC3 lipidation. Analysis of brain sections confirmed increased apoptosis and abnormal regulation of autophagy in the CNS of collagen VI-deficient animals. To investigate the in vivo physiological consequences of these CNS defects, we carried out functional studies and found that motor and memory task performances were impaired in aged Col6a1-/-mice. These findings indicate that lack of collagen VI leads to spontaneous apoptosis and defective autophagy in neural cells, and point at a protective role for this ECM protein in the CNS during physiological aging.

Reactivation of autophagy by spermidine ameliorates the myopathic defects of collagen VI-null mice.

Autophagy is a self-degradative process responsible for the clearance of damaged or unnecessary cellular components. We have previously found that persistence of dysfunctional organelles due to autophagy failure is a key event in the pathogenesis of COL6/collagen VI-related myopathies, and have demonstrated that reactivation of a proper autophagic flux rescues the muscle defects of Col6a1-null (col6a1(-/-)) mice. Here we show that treatment with spermidine, a naturally occurring nontoxic autophagy inducer, is beneficial for col6a1(-/-) mice. Systemic administration of spermidine in col6a1(-/-) mice reactivated autophagy in a dose-dependent manner, leading to a concurrent amelioration of the histological and ultrastructural muscle defects. The beneficial effects of spermidine, together with its being easy to administer and the lack of overt side effects, open the field for the design of novel nutraceutical strategies for the treatment of muscle diseases characterized by autophagy impairment.

Aggresome-Autophagy Involvement in a Sarcopenic Patient with Rigid Spine Syndrome and a p.C150R Mutation in FHL1 Gene.

The four-and-half LIM domain protein 1 (FHL1) is highly expressed in skeletal and cardiac muscle. Mutations of the FHL1 gene have been associated with diverse chronic myopathies including reducing body myopathy, rigid spine syndrome (RSS), and Emery-Dreifuss muscular dystrophy. We investigated a family with a mutation (p.C150R) in the second LIM domain of FHL1. In this family, a brother and a sister were affected by RSS, and their mother had mild lower limbs weakness. The 34-year-old female had an early and progressive rigidity of the cervical spine and severe respiratory insufficiency. Muscle mass evaluated by DXA was markedly reduced, while fat mass was increased to 40%. CT scan showed an almost complete substitution of muscle by fibro-adipose tissue. Muscle biopsy showed accumulation of FHL1 throughout the cytoplasm and around myonuclei into multiprotein aggregates with aggresome/autophagy features as indicated by ubiquitin, p62, and LC3 labeling. DNA deposits, not associated with nuclear lamina components and histones, were also detected in the aggregates, suggesting nuclear degradation. Ultrastructural analysis showed the presence of dysmorphic nuclei, accumulation of tubulofilamentous and granular material, and perinuclear accumulation of autophagic vacuoles. These data point to involvement of the aggresome-autophagy pathway in the pathophysiological mechanism underlying the muscle pathology of FHL1 C150R mutation.

Zebrafish ambra1a and ambra1b knockdown impairs skeletal muscle development.

The essential role of autophagy in muscle homeostasis has been clearly demonstrated by phenotype analysis of mice with muscle-specific inactivation of genes encoding autophagy-related proteins. Ambra1 is a key component of the Beclin 1 complex and, in zebrafish, it is encoded by two paralogous genes, ambra1a and ambra1b, both required for normal embryogenesis and larval development. In this study we focused on the function of Ambra1, a positive regulator of the autophagic process, during skeletal muscle development by means of morpholino (MO)-mediated knockdown and compared the phenotype of zebrafish Ambra1-depleted embryos with that of Ambra1gt/gt mouse embryos. Morphological analysis of zebrafish morphant embryos revealed that silencing of ambra1 impairs locomotor activity and muscle development, as well as myoD1 expression. Skeletal muscles in ATG-morphant embryos displayed severe histopathological changes and contained only small areas of organized myofibrils that were widely dispersed throughout the cell. Double knockdown of ambra1a and ambra1b resulted in a more severe phenotype whereas defects were much less evident in splice-morphants. The morphants phenotypes were effectively rescued by co-injection with human AMBRA1 mRNA. Together, these results indicate that ambra1a and ambra1b are required for the correct development and morphogenesis of skeletal muscle.

Physical exercise stimulates autophagy in normal skeletal muscles but is detrimental for collagen VI-deficient muscles.

Autophagy is a catabolic process that provides the degradation of altered/damaged organelles through the fusion between autophagosomes and lysosomes. Proper regulation of the autophagic flux is fundamental for the homeostasis of skeletal muscles in physiological conditions and in response to stress. Defective as well as excessive autophagy is detrimental for muscle health and has a pathogenic role in several forms of muscle diseases. Recently, we found that defective activation of the autophagic machinery plays a key role in the pathogenesis of muscular dystrophies linked to collagen VI. Impairment of the autophagic flux in collagen VI null (Col6a1­/­) mice causes accumulation of dysfunctional mitochondria and altered sarcoplasmic reticulum, leading to apoptosis and degeneration of muscle fibers. Here we show that physical exercise activates autophagy in skeletal muscles. Notably, physical training exacerbated the dystrophic phenotype of Col6a1­/­ mice, where autophagy flux is compromised. Autophagy was not induced in Col6a1­/­ muscles after either acute or prolonged exercise, and this led to a marked increase of muscle wasting and apoptosis. These findings indicate that proper activation of autophagy is important for muscle homeostasis during physical activity.